Project Summary Head and neck squamous cell carcinoma (HNSCC) is the most common cancer type initiated from stratified epithelium of oral and maxillofacial region. Despite great effort to characterize HNSCC via high throughput multi-omics approaches, significant improvements in patient prognosis are yet to be made. This is largely due to incomplete understanding of how cancer-associated lesions reprogram epithelial cells to expand and transform, at cellular and molecular levels. Stratified epithelium is a tissue characterized by (a) highly proliferative basal progenitor cells, (b) progenitor differentiation which is accompanied by complete loss of mitotic potential, and (c) stem cell fate choice between the proliferative progenitor and differentiated postmitotic states. As such, growth rate of stratified epithelia is controlled by balancing rates of renewal and differentiation. In my recently published work, I found that oncogenic activation of Pik3ca, the most commonly mutated oncogene in HNSCC, results in growth disadvantage in stratified epithelia. Using direct measure of cell fate choice in vivo, I found that oncogenic PI3K signaling induces differentiation. This serves to counterbalance accelerated cell cycle independently of senescence or apoptosis, and acts as a dominant cellular mechanism to restrict clonal expansion. Building on that study, my current proposal will test the hypothesis that genetic lesions and niche factors found in PI3K mutant HNSCC promote tumorigenesis by overcoming oncogene- induced differentiation. In my preliminary work I used multiple rounds of genetic screens in vivo to identify renewal promoters among ~500 patient-derived lesions associated with PIK3CA mutations. Through this large- scale effort I identified the minimal combination of lesions required to overcome PI3K induced differentiation and initiate HNSCC formation. Among these lesions, loss of Trp53 acts as a primary renewal promoter. Using a canonical p53 function deficient Trp533KR/3KR knock-in animal, I found that Trp53 loss drives HNSCC by promoting renewal, and independent of cell cycle arrest, senescence and apoptosis. By comparing chromosome accessibility between WT, Trp533KR/3KR and Trp53-/- epithelium I found that p53 can transcriptionally suppress key renewal genes, and I propose to study the importance of these genes for p53- mediated progenitor differentiation. In parallel, I identified secretory factors specifically expressed in HNSCC progenitor niche. I used a genetic screen to test their potential to overcome PI3K induced differentiation. With a novel intra-placenta lentivirus injection strategy, I am able to infect a spectrum of stromal cells present in niche of stratified epithelium. Thus, I will functional test key pro-renewal factors in stromal cells during HNSCC initiation to uncover the underlying molecular pathways mediating epithelial renewal. In summary, my preliminary data identify key genetic lesions and secreted niche factors that may drive HNSCC formation by overcoming PI3K induced differentiation. My proposal will continue to test this model, with the ultimate goal of uncovering molecular mechanism of how PI3K induced differentiation barrier is breached in HNSCCs.